The present invention relates to an avidin-type molecule which is modified at the binding-site tyrosine residue, known to be critical to the binding of biotin. The modified avidin is still capable of binding biotin or a biotinylated ligand under specific conditions, but upon altering these conditions, for example, high pH or competition with biotin, the bound biotin moiety or biotinylated ligand is removed. The invention thus provides a reversible form of avidin for use in avidin-biotin technology, thus "correcting" one of the major disadvantages of the avidin molecule for various applicative purposes, i.e., the extreme denaturing conditions required to disrupt the avidin-biotin complex. These drastic conditions necessary to dissociate the avidin-biotin complex usually inactivate irreversibly the biological activity of the biotinylated component, thus rendering it unsuitable for subsequent use.
Avidin (from egg-white) and streptavidin (from Streptomyces avidinii) are two related proteins that bind biotin with similar dissociation constants of about 10.sup.-15 M (Green, 1975). In addition to the binding of biotin, many of their physical properties are quite similar. Both, for example, are constructed of four non-covalently attached identical subunits, each of which bears a single biotin-binding site. The subunit M.sub.r values are also very similar. Moreover, several short stretches in the sequences of the two proteins are preserved, particularly to Trp-Lys stretches that occur at approximately similar positions (Argarana et al., 1986). We have previously shown (Gitlin et al., 1987, 1988a) that certain lysine and tryptophan residues are involved in the biotin binding in both proteins (Gitlin et al., 1988b). More recently, it was shown that both avidin and streptavidin exhibit the same three dimensional fold, and the most of the binding site residues are identical or similar (Weber et al., 1989). The binding site geometry and bonds formed between both proteins with the biotin molecule are indeed very similar.
Despite these similarities, several differences exist between the two proteins. Avidin is a disulphide-bridged glycoprotein containing two methionine residues, whereas streptavidin is not glycosylated and is devoid of sulphur-containing amino acid side chains. Another significant difference is in the tyrosine content. Avidin has only one tyrosine residue (Tyr-33), whereas streptavidin has six tyrosine residues at positions 22, 43, 54, 60, 83 and 96. Interestingly, the single tyrosine residue of a avidin is located in a region which contains a sequence identical with that of one of the streptavidin tyrosine residues (Tyr-43 in the stretch Thr-Gly-Thr-Tyr). This tyrosine residue occupies a prominent position in the biotin-binding site and the chemical modification of the tyrosine hydroxyl group leads to irreversible inactivation of the avidin molecule (Gitlin et. al., 1990).
Each avidin monomer binds one molecule of biotin. The unique feature of this binding, of course, is the strength and specificity of formation of the avidin-biotin complex. The resultant affinity constant, estimated at 1.6.times.10.sup.15 M.sup.-1 for avidin and 2.5.times.10.sup.13 M.sup.-1 for streptavidin (Green, 1990), is the highest known for a protein and an organic ligand. It is so strong that biotin cannot be released from the binding site, even when subjected to a variety of drastic conditions such as high concentrations of denaturing agents at room temperature, e.g., 6M guanidinium hydrochloride, 3M guanidinium thiocyanate, 8M urea, 10% .beta.-mercaptoethanol or 10% sodium dodecyl sulfate. Under combined treatment with guanidinium hydrochloride at low pH (1.5) or upon heating (&gt;70.degree. C.) in the presence of denaturing agents or detergents, the protein is denatured, and biotin is dislodged from the disrupted binding site.
Avidin recognizes biotin mainly at the ureido (urea-like) ring of the molecule. The interaction between the binding site of avidin with the sulfur-containing ring of the valeric acid side chain of the vitamin is of much lower strength. The relatively weak interaction between the carboxy-containing side chain of biotin and avidin means that the former can be modified chemically and attached to a wide variety of biologically active material; the biotin moiety of the resultant derivative or conjugate is still available for interaction with avidin. In turn, the avidin can be derivatized with many other molecules, notably "probes" or reporter groups of different types.
This is the crux of avidin-biotin technology (Wilchek and Bayer, 1990). Thus, a biologically active target molecule in an experimental system can be "labeled" with its biotinylated counterpart (a binder), and the product can then be subjected to interaction with avidin, either derivatized or conjugated with an appropriate probe.
The use of the egg-white avidin in the avidin-biotin technology is sometimes restricted due to the high basicity (pI.about.10.5) and presence of sugar moieties on the avidin molecule, which may lead to nonspecific or otherwise undesired reactions. In recent years, the bacterial protein streptavidin, has largely replaced egg-white avidin for most applications in avidin-biotin technology. However, the problems with streptavidin (high cost and biotin-independent cell binding) has prompted renewed interest in egg-white avidin as the standard for avidin-biotin technology. For this purpose, modified avidins exhibiting improved molecular characteristics both over the native protein (and previous derivatives thereof) as well as over streptavidin, have been prepared, such as N-acyl avidins, e.g., N-formyl, N-acetyl and N-succinyl avidins. These derivatives of avidin reduce the charge of the protein, but they are all prepared via covalent attachment to the available lysines of avidin, and the consequent blocking of the free amino groups hinders subsequent preparation of other types of conjugates (notably protein-protein conjugates such as avidin-labeled enzymes) which are often prepared by crosslinking via lysine residues using bifunctional reagents (e.g., glutaraldehyde).
A more useful and effective alternative to lysine modification is the modification via arginines. In this case, the pI of the protein is efficiently reduced and the lysines are still available for subsequent interaction. Two different derivatives of avidin which are modified in this manner are commercially available. One, ExtrAvidin.RTM., can be obtained in various functionally derivatized or conjugated forms from Sigma Chemical Company (St. Louis, Mo.). A second, NeutraLite Avidin.TM. (a product of Belovo Chemicals, Gastogne, Belgium) is additionally modified and can be purchased in bulk quantities.
Although the reduction of the pI of egg-white avidin solves one of the problems, the presence of the oligosaccharide residue remains a serious source of nonspecific (biotin-independent) interaction which restricts its application. The return of egg-white avidin as the standard for avidin-biotin technology has been contingent upon the removal of its sugars.
The possibilities for removing a sugar from a glycoprotein are quite limited; it is possible to do so either chemically or enzymatically. The chemical methods currently available, e.g., using HF or periodate oxidation, are either destructive or inefficient. The well known enzymatic method, which employs N-glycanase (Tarentino et al., 1984), is usually very expensive and not very effective for avidin when conventional methodology is used. Eventually, a viable procedure for deglycosylation was established and the resultant product was subsequently modified chemically via the arginines and is known under the trade mark NeutraLite Avidin.TM. (Belovo Chemicals).
In spite of these improvements, one of the main problems in the several applications of the avidin-biotin technology is the lack of reversibility of the binding and the difficulty of separating the avidin and the biotin moieties at the end of the process, without denaturation of the avidin or damaging or inactivating the biological material which had been attached via the biotin bridge. Alternatively, it would be advantageous (particularly for industrial use) to remove damaged or inactivated material from an avidin column, thus reconstituting the column for attachment of the new sample of biotinylated component.
It is an object of the present invention to provide modified avidins which are still biotin-binding and can be advantageously used in methods employing the avidin-biotin technology in which reversibility of the method is desired or is an advantage.
Nitration of tyrosine residues in model peptides and proteins using tetranitromethane has been described (Riordan et al., 1966; Sokolovsky et al., 1967). In a previous work of the present inventors (Gitlin et al., 1989), a nitrotyrosine derivative of avidin was prepared by nitration of egg-white avidin dissolved in 9M-urea with tetranitromethane (TNM). The resultant nitro-avidin preparation was inactive, i.e. it failed to bind biotin, because the nitration was carried out on a denatured form of avidin (in the presence of urea). The nitro-avidin thus prepared is entirely inadequate for use in avidin-biotin technology.
.sup.125 I-labelled avidin and streptavidin have been prepared for analytical purposes. The single tyrosine residue of each avidin subunit is not readily accessible to iodination. Avidin is rendered susceptible to iodination (chloramine T method) by the introduction of 3-(p-hydroxyphenyl)propionyl groups and thus .sup.125 I-labelled avidin containing said groups was prepared. .sup.125 I-labelled Bolton-hunter reagent can also be employed to label avidin (Finn and Hofmann, 1985). .sup.125 I-streptavidin was produced by iodination of streptavidin with Na.sup.125 I using the iodogen method (Suter et al., 1988). Unlabelled iodinated avidin and streptavidin have not been described heretofore.
Azotization and amination of tyrosine residues in model peptides and proteins, e.g. ribonuclease A, has been previously described (Gorecki et al., 1971; Sokolovsky et al, 1967).